Evaluating Feruloyl Esterase—Xylanase Synergism For

agronomy

Article Evaluating Feruloyl Esterase—Xylanase Synergism for Hydroxycinnamic Acid and Xylo-Oligosaccharide Production from Untreated, Hydrothermally Pre-Treated and Dilute-Acid Pre-Treated Corn Cobs

Lithalethu Mkabayi 1 , Samkelo Malgas 1 , Brendan S. Wilhelmi 2 and Brett I. Pletschke 1,*

1 Enzyme Science Programme (ESP), Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, Eastern Cape 6140, South Africa; [email protected] (L.M.); [email protected] (S.M.) 2 Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, Eastern Cape 6140, South Africa; [email protected] * Correspondence: [email protected]; Tel.: +27-46-6038081

Received: 4 April 2020; Accepted: 30 April 2020; Published: 13 May 2020

Abstract: Agricultural residues are considered the most promising option as a renewable feedstock for biofuel and high valued-added chemical production due to their availability and low cost. The efficient enzymatic hydrolysis of agricultural residues into value-added products such as sugars and hydroxycinnamic acids is a challenge because of the recalcitrant properties of the native biomass. Development of synergistic enzyme cocktails is required to overcome biomass residue recalcitrance, and achieve high yields of potential value-added products. In this study, the synergistic action of two termite metagenome-derived feruloyl esterases (FAE5 and FAE6), and an endo-xylanase (Xyn11) from Thermomyces lanuginosus, was optimized using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate) and then applied to 1% (w/v) corn cobs for the efficient production of xylo-oligosaccharides (XOS) and hydroxycinnamic acids. The enzyme combination of 66% Xyn11 and 33% FAE5 or FAE6 (protein loading) produced the highest amounts of XOS, ferulic acid, and p-coumaric acid from untreated, hydrothermal, and acid pre-treated corn cobs. The combination of 66% Xyn11 and 33% FAE6 displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to Xyn11 alone), respectively. The hydrolysis product profiles revealed that xylobiose was the dominant XOS produced from untreated and pre-treated corn cobs. These results demonstrated that the efficient production of hydroxycinnamic acids and XOS from agricultural residues for industrial applications can be achieved through the synergistic action of FAE5 or FAE6 and Xyn11.

Keywords: Ferulic acid; Feruloyl esterase; Xylanase; Synergy; Xylo-oligosaccharides

1. Introduction Lignocellulosic biomass is widely considered one of the most promising low-cost feedstocks for the production of renewable energy and value-added chemicals. It is primarily composed of three major components, namely: cellulose (40–60%), hemicellulose (20–40%), and lignin (10–25%) [1]. Waste agricultural residues generated during crop harvesting and processing (e.g., rice straw, wheat straw, corn stover, corn cobs, sugarcane bagasse, sorghum bagasse, etc.) are renewable biomass resources that are readily available and inexpensive [2]. A signiﬁcant amount of research has focused on developing environmentally friendly methods of utilising lignocellulosic biomass. In this regard, enzymatic conversion has emerged as a major technological platform that oﬀers several advantages

Agronomy 2020, 10, 688; doi:10.3390/agronomy10050688 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 688 2 of 14 such as environmental benefits and lower energy costs, with no formation of undesirable by-products. Due to the complexity and heterogeneity in the structure of biomass, its enzymatic conversion requires the synergistic cooperation of several enzymes [3]. A detailed understanding of this synergistic cooperation is key in the development of enzyme cocktails for the optimal production of value-added chemicals from lignocellulosic biomass. Feruloyl esterases (FAEs, EC 3.1.1.73) and endo-xylanases (EC 3.2.1.8) are essential enzymes for the degradation of xylan, the most abundant hemicellulose in biomass. FAEs catalyze the cleavage of covalent ester linkages between hydroxycinnamic acids and polysaccharides, releasing ferulic acid (FA) and p-coumaric acid (p-CA) from lignocellulosic biomass [4,5]. FA, the most abundant hydroxycinnamic acid, is usually esterified at the C-5 hydroxy group of the arabinofuranosyl units of arabinoxylan of commelinid plants [6]. Hydroxycinnamic acids are used in the food industry as precursors for vanillin, p-hydroxybenzoic acid and 4-vinylphenol production, and as food preservatives because of their antimicrobial properties [7], while in the pharmaceutical industry, they can be used for their antioxidant and anti-inflammatory properties [8–11]. Application of FAEs not only releases hydroxycinnamic acid, but also decreases biomass recalcitrance, making it more accessible for further hydrolysis by carbohydrate-active enzymes [11]. Endo-xylanases are essential in degrading xylan as they catalyze the random cleavage of β-1,4-D-xylosidic linkages, generating xylo-oligosaccharides (XOS) [12]. The enzymes have been classified into glycoside hydrolase (GH) families 5, 8, 10, 11, 30, 43, 62 and 98, with GH10 and 11 being the two families that have been extensively characterized (www.cazy.org). Endo-xylanase generated XOS from agricultural residues are used in food, feed, and pharmaceutical industries due to their prebiotic and antioxidant activity [13,14]. FAEs exhibit a synergistic interaction with xylanases during the hydrolysis of a range of lignocellulosic substrates, which is demonstrated by improved yields in the production of XOS and FA. Xylanases generate ferulated XOS, which become preferred substrates for FAEs to cleave ester bonds from, liberating FA as a product [15]. The removal of FA then increases the accessibility of xylanases to XOS for further hydrolysis into shorter XOS and/or xylose. Studies have reported significant increases in the amount of FA released from arabinoxylan after the enzymatic hydrolysis by FAEs in the presence of xylanases [16–18]. Although FAEs from different microorganisms have been used for the co-production of FA and XOS, some of these FAEs show limited hydrolysis efficiencies. Therefore, novel FAEs with exceptional catalytic properties are still required for the formulation of more efficient enzyme cocktails. Rashamuse and co-workers [19] functionally screened novel FAEs from the hindgut prokaryotic symbionts of Trinervitermes trinervoides termite species, but the application of these enzymes in degrading agricultural residues has not yet been explored. In this study, the synergistic action of two new termite metagenome derived FAEs (FAE5 and FAE6) and a GH11 xylanase from Thermomyces lanuginosus was optimized on insoluble wheat arabinoxylan (model substrate) and then applied to corn cobs (a natural substrate) for the production of XOS and hydroxycinnamic acids. The study presented demonstrated that high quantities of XOS, p-CA and FA were generated from corn cobs as a result of synergistic interactions between Xyn11 and FAE5 or FAE6.

2. Materials and Methods

2.1. Chemicals, Substrates and Enzymes Escherichia coli BL21 (DE3) was used for the expression of fae5 and fae6 genes harboured in pET28 plasmids. The cells were cultured in Luria-Bertani (LB) medium containing 50 µg/mL kanamycin at 37 ◦C. The induction was conducted by following the method described previously [20]. Enzymes were puriﬁed using 5 mL Ni-NTA Superﬂow Cartridges (QIAGEN®), purchased from QIAGEN (Germany), as per the manufacturer’s instructions. Xylanase (Xyn11) from Thermomyces lanuginosus and ethyl ferulate (EFA) were purchased from Sigma Aldrich (South Africa). Insoluble wheat arabinoxylan (WAX) was purchased from Megazyme™ (Ireland). Agronomy 2020, 10, 688 3 of 14

2.2. Pre-Treatment of Corn Cobs Milled corn cobs (CC) was treated using hydrothermal pre-treatment or dilute acid pre-treatment. A total of 10 g of biomass was suspended in Milli-Q water (for hydrothermal treatment) or in a 0.5% (w/w) sulphuric acid solution (for dilute acid treatment) (solid: liquid ratio of 1:10) and autoclaved for 20 min at 121 ◦C. The pre-treated CC slurry was ﬁltered, followed by washing the solids repeatedly with Milli-Q water and then oven drying to constant weight at 50 ◦C for 48 h.

2.3. Chemical Characterization of CC The total carbohydrates of CC were determined in triplicate by using a modified sulphuric acid method described previously [21]. Briefly, 300 mg of CC (untreated and pre-treated) was hydrolyzed with 72% (v/v) sulphuric acid at 30 ◦C for 1 h, diluted to 3% (v/v) sulphuric acid and autoclaved to solubilize the carbohydrate fraction. Following the hydrolysis, fractions were filtered to remove the insoluble lignin from the solution. Determination of the alkali-extractable hydroxycinnamic acid content was carried out by treating 10 mg of biomass with 1 M NaOH solution for 24 h at room temperature and in the dark. The liquors obtained from alkaline treatments were separated from the solid fraction by centrifugation at 16,000 g × for 5 min. The liquors were neutralized by 2 volumes of 1 M HCl and analyzed by HPLC as described in Section 2.4. The morphological structure of untreated and pre-treated biomass was analyzed with a scanning electron microscope (SEM), JOEL JSM 840. CC samples were mounted on a metal stub with adhesive tape and coated with a thin layer of gold prior to SEM analysis. In order to detect changes in functional groups, FTIR analysis of untreated and pre-treated CC was conducted by loading a few milligrams of pulverized samples in the universal ATR of a Spectrum 100 FT-IR spectrometer system (Perkin Elmer, Wellesley, MA). FT-IR spectra were recorded in quadruple at 1 1 a range of 650–4000 cm− with a resolution of 4 cm− .

2.4. Determination of Enzyme Activities and Protein Concentration For feruloyl esterase activity assay, ethyl ferulate (EFA) was used as substrate. The reaction (1 mL) was carried out in sodium phosphate buffer (50 mM, pH 7.4) that contained 1 mM EFA. The reaction was initiated by the addition of a diluted enzyme solution. After 15 min incubation at 40 ◦C, the enzymatic activity was terminated by incubation at 100 ◦C for 5 min. The released FA from the substrate was quantified using a Shimadzu HPLC system (Shimadzu Corp, Japan) equipped with a diode array detector (DAD), where the chromatographic separation was achieved using a Phenomenex® C18 5 µm (150 4.6 mm) LC column (Phenomenex, United States of America). Ambient conditions were used × for analysis. The mobile phase A was 0.01 M phosphoric acid and the mobile phase B consisted of HPLC grade acetonitrile. The isocratic mobile phase consisted of A: 70% and B: 30% and ran for 10 min at a flow rate of 0.8 mL/min. The injected volume was 10 µL and the UV absorption of the effluent was monitored at 320 nm. One unit of enzyme activity was defined as the amount of enzyme that released 1 µmol of ferulic acid per min under standard conditions. For xylanase activity assay, 10 mg/mL of insoluble wheat arabinoxylan (WAX) was used as a substrate. The reaction mixture consisted of 300 µL of 1.33% (w/v) substrate dissolved in 50 mM sodium phosphate buffer (pH 7.4) and 100 µL of diluted enzyme. After 15 min incubation at 40 ◦C, the enzymatic activity was terminated by incubation at 100 ◦C for 5 min, followed by centrifugation at 16,000 g for 5 min. The concentrations of reducing sugar were measured using 3,5-dinitrosalicylic × acid (DNS) method described previously [22]. Briefly, 150 µL of the sample was mixed with 300 µL of DNS followed by boiling for 5 min. One unit of enzyme activity was defined as the amount of enzyme that released 1 µmol of reducing sugars per min. Reducing sugars were estimated using a xylose standard curve. The yield of reducing sugars was determined using the following equation:

XOS yield (%) = ((XOS released 0.88)/ xylan content) 100 (1) × × Agronomy 2020, 10, 688 4 of 14

The Bradford method was used to determine the protein concentration of the enzymes [23]. A protein standard curve was constructed using bovine serum albumin as a suitable standard.

2.5. Synergy Studies In order to study the synergistic interactions on agricultural residues, the FAE and Xyn11 combinations were first tested on WAX for the release of FA and XOS. Enzyme loadings for all single enzymes and all enzyme combinations were kept at a total protein loading of 4 mg protein per g of WAX and at 8 mg per g of CC. For combination experiments, an enzyme mixture consisting of Xyn11 and FAE in a 66:33% protein ratio was used. The enzymatic hydrolysis was performed at a substrate loading of 0.5% (w/v) for WAX and 1% (w/v) for CC in 50 mM phosphate buffer (pH 7.4) in a total volume of 400 µL. The reaction mixture was incubated at 40◦C with agitation at 25 rpm for 24 h and terminated by boiling at 100 ◦C for 5 min. Hydrolysis controls included reactions without the addition of enzyme or substrate. All the experiments were performed in triplicate. The amount of FA and p-CA released was determined using the HPLC method described in Section 2.4, while reducing sugars were measured using the DNS method and XOS were quantified using the HPLC-RID method described in Section 2.7.

2.6. Determination of Xylo-Oligosaccharides Pattern Profiles In order to determine the hydrolysis product profiles from synergy studies, XOS were analyzed by thin-layer chromatography (TLC). Five µL of hydrolysate sample and a mixture of XOS standards were applied on a silica gel 60 F254 plate (Merck, Darmstadt, Germany). The migration was repeated twice using a mobile phase consisting of 1-butanol, acetic acid and water in a 2:1:1 ratio, respectively. The plate was then submerged in Molisch’s Reagent (0.3% (w/v) α-naphthol dissolved in methanol and sulphuric acid in a 95:5 ratio (v/v), respectively). The spots corresponding to the different XOS were visualized by heating the plate in the oven at 110 ◦C for 10 min. The XOS were quantified by a Shimadzu HPLC system (Shimadzu Corp, Japan) equipped with a refractive index detector (RID) using a CarboSep CHO 411 column (Anatech, South Africa) with water as the mobile phase in isocratic mode. The column oven was set at 80 ◦C and separation was performed within 35 min at a flow rate of 0.3 mL per min. An injection volume of 20 µL was employed for all samples and XOS standards.

2.7. Statistical Analysis All statistical analyses were performed on GraphPad Prism 6.0 software using the t-test. A p-value of less than 0.05 was considered to indicate statistically signiﬁcant diﬀerences between compared data sets.

3. Results

3.1. Chemical Characterization of CC The main hurdle in utilising lignocellulosic biomass lies in its recalcitrant nature due to the complexity of its biomass structure. Thus, to increase the enzymatic hydrolysis of biomass, various pre-treatment strategies are usually required. However, some of the pre-treatment methods are known for easily solubilizing some of the polysaccharides, mostly hemicellulose [24]. It is, therefore, critical to perform pre-treatment under conditions that lead to a recovery of the biomass components in a re-usable form while increasing the enzymatic digestibility. In this study, hydrothermal and dilute acid pre-treatment strategies were selected. An extremely low thermo-chemical pre-treatment severity was applied in order to preserve hemicellulose and hydroxycinnamic content in the solid fraction. In order to determine the eﬀect and eﬃciency of the pre-treatments, the surface morphology of CC was visualized with SEM. Figure1 shows the SEM micrographs of untreated and pre-treated CC. Agronomy 2020, 10, 688 5 of 14 Agronomy 2020, 10, x FOR PEER REVIEW 5 of 14

(a) (b) (c)

FigureFigure 1. 1.MorphologicalMorphological study study of corn of corncobs cobs(CC) (CC)by SEM. by SEM.SEM SEMmicrographs micrographs of (a) ofuntreated, (a) untreated, (b) (b) hydrothermal treated, and (c) acid-treated at a 2k magniﬁcation. hydrothermal treated, and (c) acid-treated at a 2k× magnification.×

HereHere it can it can be beseen seen that that the the untreated untreated sample sample appears appears to topossess possess a compact a compact structure. structure. In Incontrast, contrast, thethe micrographs micrographs of of pre-treated pre-treated CCCC samplessamples display displa distortedy distorted and and fragmented fragmented structures structures on the on surface. the surface.The structural The structural changes changes due to pre-treatmentdue to pre-treatmen mightt increase might increase the surface the areasurface of pre-treated area of pre-treated CC, which CC,could which lead could to an lead enhanced to an enhanced enzymatic enzymatic degradability. degradability. ToTo determine determine the the recoverable recoverable sugars sugars and and hydr hydroxycinnamicoxycinnamic acids acids after after pre-treatment, pre-treatment, the the chemicalchemical composition composition of ofthe the untreated untreated and and pre-tr pre-treatedeated CC CC was was also also analyzed analyzed (Table (Table 1).1 ).For For noting, noting, thethe composition composition of ofother other CC CC components components such such as aslignin lignin was was omitted, omitted, and and attention attention was was focused focused on on glucan,glucan, xylan, xylan, arabinan, arabinan, and and hydroxycinnamic hydroxycinnamic acids. acids. The content The content of glucan, of glucan, xylan, xylan, and arabinan and arabinan was slightlywas slightly higher higherin the pre-treated in the pre-treated biomass biomass compared compared to untreated to untreated biomass biomass samples. samples. There were There more were recoverablemore recoverable sugars in sugars the hydrothe in the hydrothermalrmal treated treated sample sample compared compared to the toacid the pre-treated acid pre-treated sample. sample. A slightA slight increase increase in FA in and FA andp-CAp-CA content content was was observed observed in the in theacid acid-treated-treated sample. sample. The The data data on onTable Table 1 1 confirmedconﬁrmed that that the the relevant relevant CC CC components components were were successfully successfully retained retained upon upon pre-treatment. pre-treatment.

Table 1. Chemical composition of untreated and pre-treated CC (on a percentage dry mass basis). Table 1. Chemical composition of untreated and pre-treated CC (on a percentage dry mass basis). Reducing Glucan a Xylan a ArabinanReducinga FA c p-CA c Glucan a Xylan a Arabinan a Sugars b FA c p-CA c Sugars b Untreated 30.86 0.90 11.46 0.48 7.79 0.63 53.00 0.49 0.61 0.012 0.63 0.026 ± ± ± ± 0.61± ± ± Hydrothermal treated± 36.03 0.13± 13.47 0.91± 11.24 0.84± 62.38 0.29 0.68 0.027 0.61± 0.024 Untreated 30.86 0.90 11.46± 0.48 ± 7.79 0.63 ± 53.00 0.49± ± 0.63 0.026± Acid-treated 32.58 0.85 12.22 0.48 8.52 0.55 59.20 0.150.012 0.68 0.027 0.67 0.013 Hydrothermal ± ± ± ± 0.68 ± ± ± Analysis method:36.03 ±a 0.13Megazyme 13.47 sugar ± 0.91 kits, b DNS 11.24 method, ± 0.84c HPLC. 62.38 The ± data 0.29 presented are averages0.61standard ± 0.024 treateddeviations of triplicates. 0.027 ± 0.68 ± Acid-treated 32.58 ± 0.85 12.22 ± 0.48 8.52 ± 0.55 59.20 ± 0.15 0.67 ± 0.013 0.027 To further investigate the chemical changes that took place during the pre-treatment of CC, Analysis method: a Megazyme sugar kits, b DNS method, c HPLC. The data presented are averages ± FTIR analysis was conducted. The spectra of untreated, hydrothermal and acid-treated CC are standard deviations of triplicates. 1 shown in Figure2. The absorption peaks at around 1730 cm − region are predominantly attributed toTo the further C=O stretchinginvestigate vibration the chemical of the changes ester that linkage took ofplace the during carboxylic the pre-treatment group of FA of and CC,p -CAFTIR of analysislignin andwas/ orconducted. hemicellulose The spectra [25]. The of spectrauntreated, of all hydrothermal samples show and this acid-treated peak suggesting CC are that shown changes in Figuredue to2. pre-treatmentThe absorption (observed peaks at around in SEM 1730 micrographs) cm−1 region did are not predominantly lead to the removal attributed of hemicellulose to the C=O stretchingand hydroxycinnamic vibration of the acids. ester Thelinkag FTIRe of data the carboxylic is in agreement group with of FA composition and p-CA of analysis lignin (Tableand/or1 ), 1 hemicelluloseas the hydrothermal [25]. The pre-treated spectra of sampleall samples displayed show strongthis peak absorption suggesting bands that around changes 1000 due cm −to andpre- in 1 treatmentthe region (observed 3500–3200 in cmSEM− (associated micrographs) with didβ-glycosidic not lead linkagesto the removal and OH of groups hemicellulose of glucose and units, hydroxycinnamicrespectively) [26 acids.]. The FTIR data is in agreement with composition analysis (Table 1), as the hydrothermal pre-treated sample displayed strong absorption bands around 1000 cm−1 and in the region 3500–3200 cm−1 (associated with β-glycosidic linkages and OH groups of glucose units, respectively) [26].

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Figure 2. FTIR spectra of untreated, hydrothermal treated and acid-treated CC solids registered in the Figure 2. FTIR spectra of1 untreated, hydrothermal treated and acid-treated CC solids registered in the range of 450–4000 cm− . range of 450–4000 cm−1. 3.2. Determination of Enzyme Specific Activities 3.2. Determination of Enzyme Specific Activities The specific activities of the enzymes used in this study were determined as detailed in Section 2.4 underThe Materials specific andactivities Methods. of the Xyn11 enzymes was used the only in th enzymeis study active were ondetermined WAX with as a detailed specific activityin Section of 15.082.4 under U/mg Materials of xylanase, and whileMethods. FAE5 Xyn11 and FAE6was the showed only enzyme activity active only on on EFA WAX with with specific a specific activities activity of of28.36 15.08 U/ mgU/mg and of 27.34xylanase, U/mg while of feruloyl FAE5 and esterase, FAE6 respectively. showed activity FAE5 only and on FAE6 EFA demonstrated with specific relativelyactivities ofsimilar 28.36 feruloyl U/mg and esterase 27.34 activities. U/mg of feruloyl esterase, respectively. FAE5 and FAE6 demonstrated relatively similar feruloyl esterase activities. 3.3. Release of XOS from Substrates by Enzymatic Hydrolysis 3.3. ReleaseIn order of toXOS evaluate from Substrates synergistic by interactions Enzymatic Hydrolysis between FAEs and Xyn11 on untreated and pre-treated CC (agriculturalIn order to residue),evaluate thesynergistic enzymes interactions were tested forbetween their ability FAEs toand release Xyn11 XOS on and untreated FA from and a model pre- treatedarabinoxylan CC (agricultural substrate (WAX). residue), A 0.5% the enzymes WAX substrate were tested loading for was their used ability for a to better release comparison XOS and with FA fromnatural a model substrates arabinoxylan since they substrate have xylan (WAX). contents A 0.5% of between WAX substrate 20–40%. Figureloading3 shows was used the productionfor a better comparisonof reducing sugarswith natural after enzymatic substrates hydrolysis since they of WAXhave (a),xylan untreated contents (b), of hydrothermal between 20–40%. treated Figure (c) and 3 showsacid-treated the production CC (d) by single of reducing or combinations sugars after of the en enzymes.zymatic hydrolysis The trend inof the WAX production (a), untreated of reducing (b), hydrothermalsugars was similar treated in all(c) substrates,and acid-treated with the CC Xyn11 (d) by tosingle FAE5 or/6 combinations combinations of releasing the enzymes. higher The reducing trend insugars the thanproduction those from of reducing the reactions sugars containing was simila individualr in all enzymes. substrates, As expected, with the FAE5 Xyn11 or FAE6to FAE5/6 alone werecombinations not able toreleasing release reducinghigher reducing sugars, sugars this was than also those indicated fromin the the reactions specific activitycontaining determination individual enzymes.study (Table As2 expected,). For hydrolysis FAE5 or ofFAE6 WAX alone (Figure were3a), no botht able FAEs to release displayed reducing synergy sugars, with this Xyn11 was also by indicatedimproving in the the release specific of activity reducing determination sugars. It was st foundudy (Table that Xyn11 2). For alone hydrolysis released of 0.89WAX mg (Figure/mL, while 3a), bothco-incubation FAEs displayed with FAE5 synergy or FAE6 with resulted Xyn11 by in 1.18improv mg/ingmL the (25.61% release increase) of reducing and1.22 sugars. mg/ mLIt was reducing found thatsugars Xyn11 (a 28.06% alone increase),released respectively.0.89 mg/mL, Thewhile hydrolysis co-incubation of untreated with FAE5 CC withor FAE6 xylanase resulted alone in and 1.18 a mg/mLcombination (25.61% of Xyn11 increase) and and FAE5 1.22 or FAE6mg/mL produced reducing reducing sugars sugars(a 28.06 at% concentrations increase), respectively. of 0.94 mg /ThemL, 1.17hydrolysis mg/mL, of and untreated 1.22 mg /CCmL, with respectively xylanase (Figure alone3 b).and The a combination hydrothermal of pre-treated Xyn11 and sample FAE5 exhibited or FAE6 produceda similar hydrolysisreducing patternsugars toat untreatedconcentrations CC, butof the0.94 amounts mg/mL, of 1.17 reducing mg/mL, sugars and released 1.22 mg/mL, in the respectivelyco-incubation (Figure sets were 3b). higher The hydrothermal (Figure3c). However, pre-treated the sample amount exhibited of reducing a similar sugars hydrolysis was significantly pattern toless untreated for acid pre-treatmentCC, but the amounts when Xyn11of reducing was applied sugars released alone (yield in the of co-incubation 28.80%), but asets combination were higher of Xyn11(Figureand 3c). FAE5However, or FAE6 the resultedamount of in reducing more reducing sugars sugars was significantly (yield of 100% less forfor bothacid combinations)pre-treatment whencompared Xyn11 to untreatedwas applied CC alone (Figure (yield3d). of The 28.80%), hydrothermally but a combination pre-treated of sampleXyn11 and exhibited FAE5 yieldsor FAE6 of resulted60.76% for in Xyn11more reducing alone, while sugars co-incubation (yield of 100% with for FAE5 both and combinations) FAE6 resulted compared in yields to ofuntreated 98.21% andCC 100%,(Figure respectively. 3d). The hydrothermally These results indicatepre-treated that sample FAEs play exhibited an important yields of role 60.76% in the for synergistic Xyn11 alone, hydrolysis while co-incubationof WAX, and the with arabinoxylan FAE5 and FAE6 contained resulted in untreatedin yields of and 98.21% pre-treated and 100%, CC samples.respectively. These results indicate that FAEs play an important role in the synergistic hydrolysis of WAX, and the arabinoxylan contained in untreated and pre-treated CC samples.

Agronomy 2020, 10, x FOR PEER REVIEW 7 of 14

Table 2. Specific activities of the enzymes assessed in this study (U/mg protein).

Substrate Enzyme Tested Xyn11 FAE5 FAE6 WAX a 15.08 Nd Nd EFA b Nd 28.36 27.34 a Analyzed by 3,5-dinitrosalicylic acid (DNS) method for xylanase activity and b analyzed by HPLC Agronomydiode2020 array, 10, 688 detector (DAD) for feruloyl esterase activity. “Nd” = not detected. 7 of 14

* * * *

(a) (b)

* * * *

FigureFigure 3.3. Release of reducing sugars during hydrolysishydrolysis of 0.5% wheat arabinoxylan (WAX) (a),), 1%1% untreateduntreated ((bb),), hydrothermallyhydrothermally pre-treatedpre-treated ((cc)) andand acidacid pre-treatedpre-treated CCCC ((dd)) byby individualindividual enzymesenzymes oror aa combinationcombination ofof 66%66% Xyn11:Xyn11: 33%33% FAE5FAE5 oror FAE6.FAE6. SubSub representsrepresents substratesubstrate control.control. StatisticalStatistical analysisanalysis waswas conductedconducted using usingt-test t-test for for improvement improvement of hydrolysis of hydrolysis with respectwith respect to reducing to reducing sugar by sugar the enzyme by the combinationsenzyme combinations compared compared to single to enzyme single (Xyn11),enzyme key:(Xyn11), * (p valuekey: * <(p0.05). value < 0.05).

Table 2. Specific activities of the enzymes assessed in this study (U/mg protein). 3.4. Release of Hydroxycinnamic Acids from Substrates by Enzymatic Hydrolysis To further Substrateunderstand the synergism Enzyme Tested between Xyn11 and FAEs, the release of hydroxycinnamic acids from substrates by individual enzymesXyn11 and their combinations FAE5 was evaluated. FAE6 Figure 4 shows a the release of FAWAX from WAX (a), untreated15.08 (b), hydrothermal Nd pre-treated (c), Ndand acid pre-treated EFA b Nd 28.36 27.34 treated CC (d). In the case of WAX (Figure 4a), a slight increase in FA release by FAE5 or FAE6 alone a b was observed.Analyzed by The 3,5-dinitrosalicylic combination acid of (DNS)Xyn11 method and FAE5 for xylanase or FAE6 activity significantly and analyzed improved by HPLC diode (P < array0.05) the detector (DAD) for feruloyl esterase activity. “Nd” = not detected. release of FA from 0.64 µg/mL and 0.65 µg/mL (individual enzyme) to 1.92 µg/mL and 2.68 µg/mL, 3.4.respectively. Release of HydroxycinnamicA similar pattern Acids was from observed Substrates for by the Enzymatic hydrolysis Hydrolysis of untreated CC, although a combination of Xyn11: FAE5 released more FA compared to Xyn11: FAE6 (Figure 4b). With respect to pre-treatedTo further CC, understand the amount the synergismof FA released between from Xyn11 the substrate and FAEs, without the release enzymatic of hydroxycinnamic hydrolysis was acids from substrates by individual enzymes and their combinations was evaluated. Figure4 shows the release of FA from WAX (a), untreated (b), hydrothermal pre-treated (c), and acid pre-treated treated CC (d). In the case of WAX (Figure4a), a slight increase in FA release by FAE5 or FAE6 alone was observed. The combination of Xyn11 and FAE5 or FAE6 significantly improved (p < 0.05) the release of FA from 0.64 µg/mL and 0.65 µg/mL (individual enzyme) to 1.92 µg/mL and 2.68 µg/mL, respectively. A similar pattern was observed for the hydrolysis of untreated CC, although a combination of Xyn11: FAE5 released more FA compared to Xyn11: FAE6 (Figure4b). With respect to pre-treated CC, the amount of Agronomy 2020, 10, 688 8 of 14 Agronomy 2020, 10, x FOR PEER REVIEW 8 of 14 enhancedFA released (Figures from the 4c,d). substrate This could without be enzymatic attributed hydrolysis to the disruption was enhanced of the (Figure close inter-component4c,d). This could associationsbe attributed between to the disruption major constituents of the close of inter-componentlignocellulose during associations the pre-treatment between major step. constituents Only Xyn11: of FAE6lignocellulose could significantly during the pre-treatmentimprove (p < step.0.05) Onlythe release Xyn11: of FAE6 FA from could both significantly pre-treated improve samples. (p < 0.05)The releasethe release of p-CA of FA from from CC both was pre-treatedalso observed samples. and is shown The release in Figure of p 5.-CA Similar from to CC the was results also presented observed inand Figure is shown 4c,d, inthe Figure pre-treated5. Similar samples to the already results showed presented increased in Figure amounts4c,d, the of pre-treatedreadily soluble samples p-CA beforealready enzymatic showed increased hydrolysis. amounts However, of readily the combination soluble p-CA of before Xyn11 enzymatic and FAE5 hydrolysis. or FAE6 However,were able theto releasecombination considerable of Xyn11 quantities and FAE5 of or p FAE6-CA compared were able toto releaseindividual considerable enzymes quantities and those of alreadyp-CA compared present andto individual soluble in enzymessubstrate andcontrols. those Interestingly, already present contrary and solubleto the FA in release substrate from controls. pre-treated Interestingly, samples, bothcontrary Xyn11: to the FAE5 FA releaseand Xyn11: from pre-treated FAE6 could samples, release both comparable Xyn11: FAE5 quantities and Xyn11: of p-CA FAE6 (Figure could release5b,c). However,comparable the quantities yields obtained of p-CA (Figure(no more5b,c). than However, 10%) were the yields much obtained less when (no morecompared than 10%)to release were efficienciesmuch less when(more comparedthan 70%) toreported release in e ffisomeciencies stud (moreies in the than literature 70%) reported [27,28]. inThese some results studies indicate in the thatliterature FAEs [acted27,28]. synergistically These results indicatewith Xyn1 that1 FAEsin the acted co-production synergistically of XOS, with FA Xyn11 and inp-CA the co-productionfrom the CC substrates.of XOS, FA and p-CA from the CC substrates.

* * * *

(a) (b)

* *

FigureFigure 4. ReleaseRelease of of ferulic ferulic acid acid during during the degradation the degradation of 0.5% WAXof 0.5% (a), 1%WAX untreated (a), 1% (b ),untreated hydrothermal (b), hydrothermalpre-treated (c) pre-treated and acid pre-treated (c) and acid CC pre-treated (d) by individual CC (d) enzymes by individual or a combination enzymes or of a 66%combination Xyn11: 33% of 66%FAE5 Xyn11: or FAE6. 33% SubFAE5 represents or FAE6. substrateSub represents control. substrate Statistical control. analysis Statistical was conducted analysis was using conductedt-test for usingimprovement t-test for ofimprovement Ferulic acid releaseof Ferulic by theacid enzyme release combinationsby the enzyme compared combinations to single compared enzyme to (FAE5 single/6), enzymekey: * (p (FAE5/6),value < 0.05). key: * (p value < 0.05).

Agronomy 2020, 10, 688 9 of 14 Agronomy 2020, 10, x FOR PEER REVIEW 9 of 14

* * * * * *

(a) (b) (c)

FigureFigure 5. 5. ReleaseRelease of pp-coumaric-coumaric acid acid during during the the de degradationgradation of 1% of 1%untreated untreated (a), hydrothermal (a), hydrothermal pre- pre-treatedtreated (b ()b and) and acid acid pre-treated pre-treated CC CC (c ()c by) by individual individual enzymes enzymes or or a acombination combination of of 66% 66% Xyn11: Xyn11: 33% 33% FAE5FAE5 or or FAE6. FAE6. Note, Note, WAX WAX was was not not assessed assessed asas therethere waswas no p-coumaric acid acid detected detected in in the the substrate. substrate. SubSub represents represents substrate substrate control.control. Statistical analys analysisis was was conducted conducted using using t-testt-test for forimprovement improvement of ofpp-coumaric-coumaric acid acid release release by by the the enzyme enzyme combinations combinations compared compared to single to single enzyme enzyme (FAE5/6), (FAE5 key:/6), * key: (p *(pvaluevalue < 0.05).< 0.05).

3.5.3.5. Determination Determination of of Hydrolysate Hydrolysate Product Product Profiles Profiles TheThe data data presented presented above above indicates indicates that that FAE5 FAE5 and and FAE6 FAE6 could could release release hydroxycinnamic hydroxycinnamic acids acids in thein presence the presence of Xyn11 of Xyn11 while while improving improving the productionthe production of reducing of reducing sugars suga fromrs from WAX, WAX, untreated untreated and pre-treatedand pre-treated CC. To CC. gain To again deeper a deeper insight insight into into these these synergistic synergistic interactions, interactions, the the product product patterns patterns of individualof individual enzymes enzymes and and their their combinations combinations were were evaluated evaluated for for the the types types of of XOS XOS generated. generated. Figure Figure6 shows6 shows the TLCthe analysisTLC analysis of XOS of released XOS released from the from hydrolysis the hydrolysis of WAX (a), of untreated WAX (a), (b), untreated hydrothermal (b), treatedhydrothermal (c) and acid-treatedtreated (c) and CC (d)acid-treated by single CC and (d) combinations by single and of enzymes.combinations It is of important enzymes. to It note is thatimportant the dark-yellow to note that colored the dark-yellow spots on thecolored plates spot represents on the plates glycerol represent which glycerol was used which as a was stabilizer used duringas a stabilizer storage ofduring the purified storage FAE5of the andpurified FAE6. FAE5 In the and case FAE6. of WAX In the hydrolysis case of WAX (Figure hydrolysis6a), individual (Figure enzymes6a), individual (lane 2) enzymes and combinations (lane 2) and (lane combinations 3 and 4) generated (lane 3 and xylobiose, 4) generated xylotetraose, xylobiose, xylopentaose,xylotetraose, andxylopentaose, xylohexaose. and The xylohexaose. hydrolysis product The hydrolysis patterns ofproduct CC (Figure patterns6b–d) of appearedCC (Figure to consist6b–d) appeared of xylobiose to (aconsist dominant of xylobiose product) and(a do smallminant quantities product) of xylotetraose.and small quantities The quantities of xylotetraose. of xylobiose The seem quantities to increase of forxylobiose the treated seem CC, to most increase especially for the fortreated enzyme CC, combinationsmost especially (lane for 3enzyme and 4). combinations Also, for acid (lane pre-treated 3 and 4). Also, for acid pre-treated CC (Figure 6d), Xyn11 alone (lane 2) produced very small quantities of CC (Figure6d), Xyn11 alone (lane 2) produced very small quantities of XOS, this was also observed in XOS, this was also observed in Figure 3b during the quantification of reducing sugars. Figure3b during the quantification of reducing sugars. We then further attempted to quantify the XOS generated using HPLC-RID. It is noteworthy We then further attempted to quantify the XOS generated using HPLC-RID. It is noteworthy that the HPLC-RID system used in this study couldn’t detect XOS of less than 0.05 mg/mL. Figure 7 that the HPLC-RID system used in this study couldn’t detect XOS of less than 0.05 mg/mL. Figure7 shows that the quantity of xylobiose produced by the combination of Xyn11 and FAE5 or FAE6 was shows that the quantity of xylobiose produced by the combination of Xyn11 and FAE5 or FAE6 was enhanced compared to Xyn11 alone, this pattern was more pronounced on untreated and pre-treated enhanced compared to Xyn11 alone, this pattern was more pronounced on untreated and pre-treated CC. High quantities of xylotetraose (0.24 mg/mL) were observed for WAX (Figure 7a), but there was CC. High quantities of xylotetraose (0.24 mg/mL) were observed for WAX (Figure7a), but there was no no significant increase between individual enzymes and their combinations. The highest quantities significantof xylobiose increase were produced between individualby enzyme enzymescombinations and theirfor hydrothermal combinations. pre-treated The highest CC (0.28 quantities mg/mL of xylobiosefor xyn11: were FAE5 produced and 0.40 by mg/mL enzyme for combinationsXyn11: FAE6) and for hydrothermalacid-treated CC pre-treated (0.34 mg/mL CC for (0.28 Xyn11: mg/ mLFAE5 for xyn11:and 0.43 FAE5 mg/mL and 0.40 for mgXyn11:/mL forFAE6). Xyn11: This FAE6) improvement and acid-treated in xylobiose CC (0.34 production mg/mL forwas Xyn11: significant FAE5 (p and < 0.430.05) mg when/mL forcompared Xyn11: to FAE6). single This enzyme improvement incubations, in xylobiosewhich resulted production in 0.14 wasmg/mL significant and 0.12 ( pmg/mL< 0.05) whenfor hydrothermal compared to and single acid-treated enzyme incubations,CC, respectively. which From resulted the results in 0.14 presented mg/mL above, and 0.12 it appears mg/mL that for hydrothermalXyn11 produced and XOS acid-treated which become CC, respectively. substrates to From FAEs the for results the removal presented of FA above, or p it-CA, appears and this that Xyn11allows produced Xyn11 to XOS further which hydrolyze become these substrates long XOS to FAEs into for the the shorter removal xylobiose. of FAor p-CA, and this allows Xyn11 to further hydrolyze these long XOS into the shorter xylobiose.

Agronomy 2020, 10, 688 10 of 14

Agronomy 2020, 10, x FOR PEER REVIEW 10 of 14

(a) (b)

Figure 6. Thin-layerFigure 6. chromatography Thin-layer chromatography (TLC) (TLC) analysis analysis ofof hydrolysis hydrolysis of 0.5% ofWAX 0.5% (a), 1% WAX untreated (a), (b 1%), untreated (b), hydrothermal pre-treated (c) and acid pre-treated CC (d) by (2) Xyn11 alone, (3) a combination of 66% hydrothermal pre-treatedXyn11: 33% FAE5, (c) and and (4) acida combination pre-treated of 66% CCXyn11: (d 33%) by FAE6. (2) Xyn11Substrate alone,without (3)enzyme a combination was of 66% Xyn11: 33% FAE5,used as and a control (4) a(1). combination A mixture of xylo-oligosa of 66%ccharides Xyn11: (X1-X6) 33% was FAE6. used as Substrate a standard. withoutArrows enzyme was indicated observed bands, albeit feint in some instances. used as a control (1). A mixture of xylo-oligosaccharides (X1-X6) was used as a standard. Arrows Agronomy 2020, 10, x FOR PEER REVIEW 11 of 14 indicated observed bands, albeit feint in some instances.

* * * * * * Oligosaccharide (mg/mL)

6 11 n y :FAE5 :FAE X 1 1 n1 y Xyn1 X

(a) (b)

* * * *

Figure 7. Xylo-oligosaccharideFigure 7. Xylo-oligosaccharide content content measured measured fromfrom the the hydrolysis hydrolysis of 0.5% of 0.5%WAX ( WAXa), 1% untreated (a), 1% untreated (b), hydrothermal pre-treated (c) and acid pre-treated CC (d) after incubation with Xyn11 alone or a b c d ( ), hydrothermalcombination pre-treated of 66% Xyn11: ( ) 33% and FAE5 acid or pre-treated FAE6. Statistical CC analysis ( ) after was incubation conducted using with t-test Xyn11 for alone or a combinationimprovement of 66% of Xyn11: hydrolysis 33% with FAE5 respect or to FAE6. xylo-oligosaccharides Statistical analysis (XOS) bywas the enzyme conducted combinations using t-test for improvementcompared of hydrolysis to single enzyme with respect(Xyn11), key: to xylo-oligosaccharides * (p value < 0.05). (XOS) by the enzyme combinations compared to single enzyme (Xyn11), key: * (p value < 0.05). 4. Discussion XOS and high value-added compounds generated from agricultural residues have great potential for application in pharmaceutical, food, and fine chemical industries. It has been reported that the total world production of corn, a key agricultural crop, was estimated to exceed 875,226,630 tons [29]. CC, a by-product of corn is an attractive agricultural residue due to its high xylan content and the fact that it’s readily available. The production of XOS could be achieved by application of chemical and hydrothermal pre-treatments. However, higher severity pre-treatment conditions can lead to increased decomposition of XOS to xylose and generation of unwanted by-products [14]. The application of low severity pre-treatment conditions to increase accessible area for enzymes, followed by enzymatic hydrolysis, is an effective method for XOS production. In this study, the synergistic interactions between two termite metagenome-derived FAEs and a xylanase, Xyn11, for the production of XOS, p-CA and FA from untreated and pre-treated CC was evaluated. WAX was used as a model substrate to optimize the enzyme cocktail as it maintains the ferulic acid cross-linkages in the native arabinoxylan. CC was subjected to two pre-treatment strategies, hydrothermal and dilute sulfuric acid pre-treatment. The results for composition analysis (Table 1) showed that there was an increase in recoverable sugars and that hydroxycinnamic acid content was maintained in pre-treated samples. However, the xylan content of all samples was much less than the values reported in the literature (28%) [30]. Biomass (morphology) characterization indicated that these changes were associated with increased surface area due to pre-treatment (Figure 1). It can be speculated that the increased sugar content of pre-treated CC samples (Table 1) may be due to these structural modifications which may include changes in lignin content. An increase in the production of

Agronomy 2020, 10, 688 11 of 14

4. Discussion XOS and high value-added compounds generated from agricultural residues have great potential for application in pharmaceutical, food, and fine chemical industries. It has been reported that the total world production of corn, a key agricultural crop, was estimated to exceed 875,226,630 tons [29]. CC, a by-product of corn is an attractive agricultural residue due to its high xylan content and the fact that it’s readily available. The production of XOS could be achieved by application of chemical and hydrothermal pre-treatments. However, higher severity pre-treatment conditions can lead to increased decomposition of XOS to xylose and generation of unwanted by-products [14]. The application of low severity pre-treatment conditions to increase accessible area for enzymes, followed by enzymatic hydrolysis, is an effective method for XOS production. In this study, the synergistic interactions between two termite metagenome-derived FAEs and a xylanase, Xyn11, for the production of XOS, p-CA and FA from untreated and pre-treated CC was evaluated. WAX was used as a model substrate to optimize the enzyme cocktail as it maintains the ferulic acid cross-linkages in the native arabinoxylan. CC was subjected to two pre-treatment strategies, hydrothermal and dilute sulfuric acid pre-treatment. The results for composition analysis (Table1) showed that there was an increase in recoverable sugars and that hydroxycinnamic acid content was maintained in pre-treated samples. However, the xylan content of all samples was much less than the values reported in the literature (28%) [30]. Biomass (morphology) characterization indicated that these changes were associated with increased surface area due to pre-treatment (Figure1). It can be speculated that the increased sugar content of pre-treated CC samples (Table1) may be due to these structural modifications which may include changes in lignin content. An increase in the production of reducing sugars was observed during enzymatic hydrolysis of pre-treated CC (Figure3), indicating the success of the pre-treatment strategies selected for this study. Regarding the enzymatic release of hydroxycinnamic acids from the substrates, the data presented above suggests that FAE5 or FAE6 could release significant amounts of FA and p-CA only in the presence of Xyn11. The inability of FAEs to release high quantities of hydroxycinnamic acids when incubated alone could be attributed to the type of bonds available on the substrate. FA or p-CA is usually found esterified to polysaccharides such as arabinoxylan and could also ether-link with lignin or dimerize with other hydroxycinnamic acid-linked polysaccharides, forming cross-linkages between these polymers. FAEs are known for specifically catalyzing the cleavage of ester bonds between hydroxycinnamic acids and polysaccharides, but not the ether-linkages. It is, therefore, possible that FAE5 and FAE6 couldn’t act on these cross-linked complex structures. Previous studies have indicated that FAEs could release FA from substrates when co-incubated with xylanase [27,31]. Zhang and co-workers [32] reported that the combination of a xylanase and AfFaeA increased the amount of FA released from steam exploded corn stalk 13-fold. It appears that FAE activity can be enhanced by the addition of a xylanase - it appears as if the xylanase generates feruloylated XOS from the xylan main chain which are easily hydrolyzed by FAE in comparison to feruloylated xylan. In turn, FAE action allows the xylanase to be able to further hydrolyze XOS with a high degree of polymerization into shorter chain XOS products. The patterns observed in the production of reducing sugars from acid pre-treated CC, presented in Figure3d, are indicative of such a relationship between xylanase and FAE. The activity of Xyn11 alone was drastically reduced, while the enzyme combinations resulted in the highest reducing sugar release. It is likely acid pre-treatment of CC generated XOS esterified with FA or p-CA, which may pose limitations on their accessibility (steric hindrance) for the Xyn11 to hydrolyze them to shorter chain XOS products. Therefore, the addition of FAEs resulted in the improvement of Xyn11 activity on these long, feruloylated XOS. There have been reports on oligosaccharides resulting from the pre-treatment of several natural substrates [33–35]. The hydrolysis patterns of individual enzymes and enzyme combinations were then also investigated. Xyn11 and a combination of Xyn11 with FAEs released XOS with a low degree of polymerization (DP 6–4 and DP 2, respectively) from WAX and released mainly xylobiose from untreated and pre-treated CC. The variations in product patterns observed could be due to the Agronomy 2020, 10, 688 12 of 14 differences in the xylan structure between WAX and CC. The yield of xylobiose from the pre-treated substrates was significantly increased by enzyme combinations compared to individual enzymes. It has been reported that xylobiose rich XOS are the preferred products for prebiotic activity [36]. The product patterns demonstrated that the Xyn11: FAEs enzyme combination (cocktail) has the potential of releasing XOS with a high xylobiose content from CC, most notably when combined with hydrothermal or dilute sulfuric acid pre-treatments.

5. Conclusions This study reported, for the ﬁrst time, the application of two termite metagenome-derived FAEs for the production of XOS and hydroxycinnamic acids from CC. The FA and/or p-CA released from WAX and CC was greatly enhanced when FAE5 and FAE6 were co-incubated with Xyn11 compared to when the enzymes were applied individually. The presence of FAEs played a major role in the production of XOS, mainly xylobiose, from untreated and pre-treated CC. This study suggests that FAEs act synergistically with Xyn11 during the enzymatic hydrolysis of CC to produce the industrially relevant products XOS and hydroxycinnamic acids.

Author Contributions: Conceptualization, L.M.; S.M.; B.S.W. and B.I.P.; methodology, L.M.; software, L.M.; validation, L.M.; S.M.; B.S.W. and B.I.P.; formal analysis, L.M.; S.M.; B.S.W. and B.I.P.; investigation, L.M.; resources, B.S.W. and B.I.P.; data curation, L.M.; writing—original draft preparation, L.M.; writing—review and editing, L.M.; S.M.; B.S.W. and B.I.P.; visualization, L.M.; supervision, B.I.P. and B.S.W.; project administration, B.I.P. and B.S.W.; funding acquisition, B.I.P. All authors have read and agree to the published version of the manuscript. Funding: The work was funded by the South African Department of Science and Technology (DST)/Council for Scientific and Industrial Research (CSIR) National Biocatalysis Initiative, National Research Foundation (NRF) of South Africa and Rhodes University. Any opinion, findings and conclusions or recommendations expressed in this material are those of the author(s) and therefore the NRF does not accept liability in regard thereto. Conflicts of Interest: The authors declare no conflict of interest.

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